为探究重组产气荚膜梭菌α毒素对家兔的安全性和免疫保护效力,本研究构建了含产气荚膜梭菌α毒素基因的重组质粒pVL1393-CPA,并通过BD BaculoGoldTM转染试剂盒转染Sf9细胞后,经噬斑纯化获得单克隆重组病毒,将其感染High 5细胞进行表达,表达的重组蛋白经SDS-PAGE和western blot检测,结果显示均获得了45 ku的目的蛋白,即为重组α毒素.将重组α毒素用0.1%甲醛溶液灭活脱毒后与ISA 206 VG佐剂乳化制备亚单位疫苗(抗原含量为20 μg/mL),以40 μg/只肌肉注射体质量1.5 kg～2.0 kg的家兔,同时设PBS对照组,注射后连续观察7 d,结果显示实验兔临床观察无异常,该疫苗对家兔的安全性良好.随后以20 μg/只肌肉注射家兔,免疫21 d后以相同剂量加强免疫一次,同时设佐剂对照组和α毒素对照组,首免21 d、二免14 d时采血分离血清,采用ELISA方法检测免疫兔血清IgG抗体效价,同时进行二免血清小鼠体内毒素中和试验以及家兔攻毒免疫保护试验,以评价该疫苗对家兔的免疫保护效力.结果显示,疫苗免疫组家兔血清IgG抗体效价比佐剂对照组和毒素对照组均极显著增高(P<0.001),二免血清对A型产气荚膜梭菌毒素小鼠体内的中和效价为32,疫苗免疫组家兔攻毒后均健活,攻毒保护率达100%,佐剂对照组和毒素对照组家兔攻毒后死亡率则为100%.上述结果表明制备的重组产气荚膜梭菌α毒素疫苗对家兔具有良好的安全性和免疫保护效力,本研究为开发其他各型梭菌亚单位疫苗提供了重要借鉴.

2021年12月,内蒙古、山西某肉牛场暴发呼吸道疾病.为了确诊病原,无菌采集发病牛血液、鼻拭子接种于MDBK细胞和Hayflick液体培养基进行病毒和细菌的分离鉴定,同时对分离培养物进行PCR鉴定.将分离毒株负染制样,在透射电镜下观察病毒形态;并对疑似支原体菌落进行吉姆萨染色、Dienes染色,观察菌体形态.根据病毒和细菌分离结果初步鉴定为多种病原混合感染所引起.从PCR检测以及电镜观察、染色结果可以得出,引起这次牛呼吸道疾病综合征的病原为牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛副流感病毒型(BPIV3)和牛支原体(Mycoplasma bovis).

以光滑型布鲁菌脂多糖(smooth lipopolysaccharide,sLPS)抗原为检测抗原,葡萄球菌蛋白A(SPA)为胶体金标记物,制备胶体金免疫层析试纸条,用于检测布鲁菌抗体.通过敏感性和特异性试验检测试纸条性能.结果显示,试纸条最低可检测血清浓度为0.1 IU/mL.试纸条批内和批间可重复性为100％,不与其他非相关疾病感染血清反应.试纸条法与试管凝集试验对牛、羊血清检测的符合率分别为95.4％和97.2％.结果表明,该试纸条在保证检测特异性的基础上,提高了对样品检测的敏感性,具有敏感、特异、简便、快速的特点,更加适合在基层推广使用.

研制抗蓖麻毒素精制免疫球蛋白,即马抗蓖麻毒素免疫球蛋白F(ab')2制品,为蓖麻毒素中毒的防治提供特效药.利用传统方法,从蓖麻籽中提取、纯化脱毒蓖麻毒素,加入适量弗氏佐剂充分乳化,免疫马匹,测定免疫马匹血清中和抗体效价并采血,分离血清,按照免疫球蛋白F(ab')2生产工艺制备治疗性抗体成品,并对成品进行中和抗体效力检测、安全性检测及免疫防治效果评价.结果 显示,纯化的蓖麻毒素抗原,经HPLC法测定纯度大于90％,腹腔注射对小鼠的半数致死量(LD50)约为6.38 μg/kg;免疫马匹血清中和抗体效价达1:64000以上时,采血并制备F(ab ')2成品,纯度在80％以上,中和抗体效价均超过6000 U/mL;安全性符合现行《中国药典》要求,对蓖麻毒素中毒动物模型有很好的免疫防治效果.本研究建立了蓖麻毒素精制免疫球蛋白制品生产工艺,制备的成品符合现行《中国药典》要求,为蓖麻毒素中毒的防治提供了新的救命药.

Accumulating evidence has shown that long noncoding RNAs (lncRNAs) are involved in several biological processes, including immune responses. However, the role of lncRNAs in antiviral innate immune responses remains largely elusive. Here, we identify an uncharacterized human lncRNA AVAN from influenza A virus (IAV) infected patients, that is significantly upregulated following RNA virus infection. During IAV infection, AVAN play an indispensable role in antiviral immune responses. In vivo, we enforced the expression of AVAN in transgenic mice or adeno-associated virus encoding AVAN delivery system and found that AVAN significantly alleviated IAV virulence and virus replication. Mechanistically, nuclear AVAN positively regulates the transcription of forkhead box O3A (FOXO3a) by associating with its promoter and inducing chromatin remodeling to promote neutrophil chemotaxis. Meanwhile, cytoplasmic AVAN binds directly to the E3 ligase TRIM25 and enhances TRIM25-mediated K63-linked ubiquitination of RIG-I, thereby promoting TRIM25- and RIG-I-mediated antiviral innate immune responses, including the induction of type I interferon and ISGs. Moreover, AVAN binds to the B Box/CCD domain of TRIM25 and 1–200nt of AVAN were the functional moieties. Collectively, our findings highlight the potential clinical implications of human lncRNA AVAN as a key positive regulator of the antiviral innate immune response and a promising target for developing broad antiviral therapeutics.

为评价金黄色葡萄球菌(S.aureus)Csa1A、Hlα和EsxA-B蛋白的免疫保护作用,本研究构建了重组质粒pGEX-4T-2-Csa1A、pET-22b-Hlα、pGEX-4T-2-EsxA-B 并分别转化大肠杆菌 BL21(DE3),经 IPTG诱导并利用相应层析柱纯化后,采用SDS-PAGE检测重组蛋白的表达.结果显示,获得了纯度较高的重组蛋白Csa1A(rCsa1A)、Hlα(rHlα)和 EsxA-B(rEsxA-B),纯度分别为89％、95％、85％,大小分别为54ku、33ku 和39ku.以纯化的rCsa1A、rHlα和rEsxA-B 60 μg/只分别免疫BALB/c小鼠,共免疫3次,每次间隔14d,采用间接ELISA法检测各组小鼠免疫后14d的抗体效价.结果显示,3种重组蛋白免疫后均能刺激小鼠产生较高效价的抗体,三免后抗体效价分别为1∶14 700(rCsa1A)、1∶16 890(rHlα)、1∶15 760(rEsxA-B).利用5型NM2株(2×107 cfu/只)、8型LZ145株(2×107cfu/只)、336型XJ44株(2×107cfu/只)3种血清型S.aureus分别经尾静脉注射攻菌,观察并统计各组小鼠的发病和死亡率,评价各重组蛋白的免疫保护率.结果显示,分别经原核表达了rCsa1A、rHlα和rEsxA-B.攻菌后对照组小鼠全部死亡;3组重组蛋白免疫组96h内死亡的小鼠在攻菌48h后出现精神沉郁,食欲不振等症状,而存活小鼠未出现临床症状.rCsa1A、rHlα和rEsxA-B免疫组小鼠提供的抵抗NM2、LZ145、XJ44菌株攻击的保护率分别为60％、60％、50％;80％、70％、70％;50％、60％、60％,其中rHlα的免疫保护效果最佳,其对3种攻毒菌株提供的平均保护率为73.3％,是较为理想的亚单位疫苗候选抗原.以上结果表明,S.aureus rHlα能够刺激免疫小鼠产生抗体,对小鼠提供不同程度的保护力.本研究为奶牛乳腺炎S.aureus亚单位疫苗的研制和应用提供参考依据.

为了评价本项目组自制的绵羊肺炎支原体鹦鹉热衣原体二联灭活疫苗的免疫效力,取制备的绵羊肺炎支原体SH-01株及鹦鹉热衣原体重组MOMP蛋白抗原等体积混合后与ISA201佐剂乳化制成灭活疫苗(至少含SH-01株2×108 CCU/mL;重组MOMP蛋白0.3 mg/mL),在配种前15d免疫健康母绵羊,免疫21d后采血分离血清分别测定抗绵羊肺炎支原体和抗鹦鹉热衣原体的特异抗体效价;免疫45 d后用绵羊肺炎支原体SH-01株和鹦鹉热衣原体CG1株进行攻毒试验.结果 显示,免疫后绵羊肺炎支原体血凝抗体效价高达1∶64,鹦鹉热衣原体ELISA抗体效价高达1:4850;绵羊肺炎支原体和鹦鹉热衣原体强毒株攻毒后保护率均可达到100％,表明此绵羊肺炎支原体鹦鹉热衣原体二联灭活疫苗具有良好的免疫保护效果.

本研究将鹦鹉热衣原体的MOMP基因经PCR扩增后与pET-22b(+)载体连接构建重组质粒,转化入E.coli,通过IPTG诱导表达MOMP蛋白,鉴定重组蛋白的表达和反应性.结果 显示,重组蛋白被成功表达,分子质量大小为40 ku,经溶解、透析纯化后,重组MOMP蛋白纯度为91％,能与鼠抗重组MOMP蛋白抗体产生特异性反应,与鹦鹉热衣原体阳性血清呈强阳性反应.重组MOMP蛋白与ISA201佐剂乳化制成鹦鹉热衣原体亚单位疫苗,免疫绵羊后抗体效价高达1∶4 222,用强毒株攻毒后保护率达100％,表明此鹦鹉热衣原体亚单位疫苗具有良好的免疫原性和免疫保护性.

Liver cancer is a common malignant tumor in China, and is the second leading cause of death of malignant tumor. Although there are many traditional methods of treatment, there is no significant improvement in the rate of clinical cure. The aim of this paper is to generate a recombinant influenza virus and evaluate its effect of intracorporeal and extracorporeal lytic tumor. Using reverse genetics (RG) technology, recombinant plasmids pFlu-CTLA4-PB1 and pFlu-CTLA4-PA were constructed by inserting heavy and light chains of CTLA4 antibodies into PR8 virus PB1 and PA gene fragments. The remaining six plasmids PB2, HA, NP, NA, M, and NS of the PR8 virus were transferred to COS-I and MDCK cells. The obtained recombinant virus, named as rFlu-muCTLA4, was identified by hemagglutination test, EID50, PCR and other methods. ELISA was used to determine the anti-CTLA4 antibody content; MTS in vitro was carried out to determine the effect of recombinant virus on mouse liver cancer H22 cells; and mouse model of liver cancer was used to evaluate the effect of recombinant virus via tumor inhibition in vivo. Data showed that the PB1 and PA fragments of the recombinant virus are the same size as expected, and the hemagglutination titer of rFlu-muCTLA4 was 29 to 210. anti-CTLA4 antibodies could be detected in 2 days of virus inoculation of chicken embryo culture and reach a peak value in 4 days. In vitro experiments showed that rFlu-muCTLA4 could specifically kill H22 cells in dose-dependent manner.Animal experiments had shown that the recombinant virus could significantly reduce the tumor volume of liver cancer in mice, increase animal survival rate and extend survival time. The results show that recombinant influenza virus rFlu-muCTLA4 expressing the immune checkpoint CTLA4 antibody can target killing liver cancer cells in vitro and in vivo, has potential to provide a novel strategy for the targeted treatment of HCC.

目的:分离、筛选柯萨奇病毒A组10型(Coxsackievirus A 10,CVA10)病毒候选疫苗株,并对其免疫原性及免疫保护性进行评价,为CVA10疫苗的研制奠定基础.方法:本研究从北京市儿童医院、军事医学研究院、北京市疾控中心共收集80个手足口病(hand foot and mouth disease,HFMD)标本,通过细胞培养法、RT-PCR的方法分离、鉴定CVA10病毒株.对病毒进行嗜斑纯化,各纯化病毒株与Al(OH)3(0.5 g/ml)佐剂混合后制备原疫苗,免疫Balb/c小鼠,收集血清.应用交叉中和实验及抗体阳转率实验筛选候选CVA10疫苗株.筛选后T9疫苗株免疫ICR乳鼠,观察ICR乳鼠的致病情况.结果:共分离出14株CVA10病毒株.交叉中和实验筛选得到4株候选疫苗株.最后阳转率实验确定T9为候选疫苗株.Al(OH)3+CVA10灭活抗原在Balb/c小鼠体内能诱生高滴度的特异性抗体,该抗体对Balb/c小鼠有保护作用.T9病毒免疫ICR乳鼠后,对乳鼠完全致死.结论:筛选1株CVA10病毒候选疫苗株,CVA10候选疫苗对ICR乳鼠动物模型有致病性.

Staphylococcal enterotoxin B (SEB) produced by the Staphylococcus aureus bacteriumis most commonly associated with food poisoning and is known to also cause toxic shock syndrome. Currently, no approved vaccine or specific drug is available to treat SEB intoxication. In this study, we fabricated dissolving microneedles (MNs) loaded with recombinant SEB (rSEB) protein, and evaluated its characteristics, including dissolution profile, protein particle size, insertion depth, antigen retention time in vivo, and skin irritation. Our results showed that rSEB protein-loaded dissolving MNs made of chondroitin sulfate (2%) and trehalose (0.8%) could easily penetrate into the mouse skin within 5 min. The rSEB particle size was unchanged before and after MN fabrication. The skin penetration depth of the MNs was 260 µm. Moreover, the MNs also significantly extended the antigen retention time in vivo. rSEB protein-loaded dissolving MNs also triggered slight erythema at the beginning of administration, but this erythema disappeared within a few hours. More importantly, we investigated the immunogenicity and protective efficacy of rSEB protein-loaded dissolving MNs. Challenge studies in mice revealed that mice in full-dose MN group had a high level of SEB specific antibody response thatprovided100% protection against a lethal SEB toxin challenge. However, there was only 60% protection observed in mice that were in the half-dose MN (dose sparing) group. We also determined the pathological alterations in the tissues of the immunized mice. Taken together, these dissolving MNs may present a promising transcutaneous immunization strategy for treating SEB intoxication.

目的:研制金黄色葡萄球菌肠毒素B(SEB)精制免疫球蛋白,即马抗SEB免疫球蛋白F(ab′)2制品,为SEB中毒的救治提供候选药.方法:利用传统的发酵和纯化工艺制备天然SEB纯品,并建立小鼠实验动物模型;利用分子克隆和蛋白质纯化技术,克隆、表达、纯化重组SEB,加入适量弗氏佐剂充分乳化,免疫马匹,测定免疫马匹血清中和抗体效价后采血,分离血清,按照成熟的生产工艺制备治疗性抗体成品;对成品进行安全性检测及免疫救治效果评价.结果:纯化的天然SEB经SDS-PAGE和HPLC法测定纯度大于90％,对小鼠腹腔注射的半数致死量为0.5μg/kg;纯化的重组SEB表达量超过2 mg/mL,经SDS-PAGE和HPLC法测定纯度大于90％,对小鼠腹腔注射的半数致死量为50μg/kg;免疫马匹血清中和抗体效价达2000 U/mL以上时,采血并制备F(ab′)2成品,经SDS-PAGE和HPLC法测定纯度在80％以上,中和抗体效价超过2500 U/mL;安全性符合现行《中国药典》要求;对SEB中毒动物模型有很好的免疫救治效果.结论:建立了SEB精制免疫球蛋白制品生产工艺,制备的成品符合现行《中国药典》要求,为SEB中毒的救治提供了候选药.

Oncolytic virotherapy is a promising strategy for the treatment of cancer. Influenza A virus has shown potential as an oncolytic agent. In this study, a recombinant PR8 influenza viral vector, called delNS1-GM-CSF, was generated with a partial deletion in NS and the granulocyte-macrophage colony-stimulating factor (GM-CSF) coding sequence inserted into the influenza nonstructural protein 1 gene. The morphological characteristics of delNS1-GM-CSF were examined. The delNS1-GM-CSF virus replicated well in various cell lines, including MDCK, A549, SMCC7721, and HepG2 cells. Moreover, selective cytotoxicity of the virus was observed in various hepatocellular carcinoma (HCC) cell lines, while no effect was demonstrated in the normal liver cell line LO2, as indicated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet assays. Importantly, using a model based on the growth of HepG2 cells as a xenograft in nude mice, it was found that a reassortant delNS1-GM-CSF virus inhibited tumor growth significantly following intratumoral injection in a dose-dependent manner. Ex vivo results showed that the tumor inhibition efficacy of delNS1-GM-CSF was observed in HCC clinical samples. Taken together, these results are the first to demonstrate that influenza A viruses may have potential as oncolytic virotherapeutic agents against HCC.

目的 建立重组金黄色葡萄球菌肠毒素B(SEB)蛋白的可溶性微针体外检测方法及体内免疫效果评价.方法 通过动态光散射(DLS)检测蛋白粒径、BCA法检测单片微针的蛋白浓度、活体成像实验检测微针皮内注射后蛋白在体内的停留时间,通过DRIZE评分评价微针注射后的皮肤情况,通过动物实验评价重组SEB蛋白可溶性微针的免疫原性及免疫保护效果.结果 DLS分析显示SEB蛋白可溶性微针负载的重组SEB蛋白粒径与重组SEB蛋白溶液中的SEB蛋白粒径无明显差别,单片微针中重组SEB蛋白含量为(13.2±1.4)μg,活体成像实验显示相对于肌肉注射,重组SEB蛋白可溶性微针皮内给药后荧光蛋白在体内的停留时间延长,动物实验显示13.0 μg的重组SEB蛋白可溶性微针即能引发免疫反应,并且在2LD50SEB攻毒剂量下能够产生很好保护效果.结论 重组SEB蛋白可溶性微针能够刺激小鼠机体产生较好的免疫原性及免疫保护效果,为发展疫苗新型免疫途径提供了新策略.

目的 构建两质粒冷适应流感病毒拯救系统,提高流感病毒的重配效率.方法 本研究通过构建克隆载体pfastbac △plhHTB,将流感病毒A/Ann Arbor/6/60 (H2N2)6个内部基因(PB2、PB1、PA、NP、M、NS)的双向转录表达元件定向克隆入此载体,获得pfastbac△plhHTBP6.同时将流感病毒A/California/7/2009 (H 1N1)表面基因(HA、NA)双向转录表达元件定向克隆入pENTR-1A质粒,获得pENTR-1AP2.将2个重组质粒共转染MDCK/COSI细胞,细胞上清接种10日龄SPF鸡胚,收取尿囊液,血凝实验筛选阳性株,并进行全面鉴定.结果 本研究利用两质粒系统成功重配甲型H1N1流感病毒株,命名为TRG A/California/07/2009(H1N1) Vca,重配病毒株传代后HA效价为28,形态符合流感病毒典型特征,大小80～120 nm,具有冷适应、温度敏感表型.结论 两质粒流感病毒拯救系统为高效重配流感疫苗株提供了新策略.

Influenza virus (IAV) infection is a major cause of severe respiratory illness that affects almost every country in the world. IAV infections result in respiratory illness and even acute lung injury and death, but the underlying mechanisms responsible for IAV pathogenesis have not yet been fully elucidated. In this study, the basic fibroblast growth factor 2 (FGF2) level was markedly increased in H1N1 virus-infected humans and mice. FGF2, which is predominately derived from epithelial cells, recruits and activates neutrophils via the FGFR2-PI3K-AKT-NFκB signaling pathway. FGF2 depletion or knockout exacerbated influenza-associated disease by impairing neutrophil recruitment and activation. More importantly, administration of the recombinant FGF2 protein significantly alleviated the severity of IAV-induced lung injury and promoted the survival of IAV-infected mice. Based on the results from experiments in which neutrophils were depleted and adoptively transferred, FGF2 protected mice against IAV infection by recruiting neutrophils. Thus, FGF2 plays a critical role in preventing IAV-induced lung injury, and FGF2 is a promising potential therapeutic target during IAV infection.

Outbreaks of hand, foot and mouth disease (HFMD), which is caused by Enterovirus 71 (EV71), have erupted in recent years. Andrographolide sulfonate (Trade name: Xiyanping injection) has been recommended to treat severe HFMD in China because of its conventional antithermic and antitoxic activities, but its actual mechanism has not been revealed clearly until now. To explore its therapeutic efficacy and mechanism, a Xiyanping injection treatment mouse model was established. Based on the therapeutic model, routine clinical parameters and histopathologic changes were investigated, in the same time, viral loads, immune cells, inflammatory molecules and cell signaling pathways were determined. Xiyanping injection treatment protected mice from lethal EV71 challenge in a therapeutic regimen-dependent manner, which may mostly depend on its direct immunomodulatory activities on neutrophil and T lymphocyte. Reduced inflammatory molecular production of neutrophil and elevated T lymphocyte activity may result from its marked inhibition of some signaling pathways. Taken together, Xiyanping injection was an effective treatment for severe HFMD by improving hosts' immunity.

目的 探讨加巴喷丁对呼吸道合胞病毒(respiratory syncytial virus,RSV)的抗病毒作用.方法 体外实验通过MTS、TCID50、qRT-PCR方法检测不同浓度加巴喷丁对RSV感染的抑制作用,包括细胞活力、病毒复制及细胞因子的变化.体内实验选取4～6周龄C57BL/6小鼠,随机分为空白对照组、RSV感染组、低剂量及高剂量加巴喷丁处理组,连续腹腔注射给药,每日观察体质量变化,HE染色观察小鼠肺部的病理变化,qRT-PCR方法检测肺部病毒载量.结果 1、2、5、10 mmol/L不同浓度加巴喷丁显著增加RSV感染A549细胞的活力;5、10 mmol/L浓度的加巴喷丁可显著降低RSV感染A549细胞的病毒载量,10 mmol/L的加巴喷丁可显著抑制病毒的复制,减少CCL3、CCL5、CXCL2及TNF-α、IL-6、IL-8等趋化因子和炎性因子表达,促进干扰素IFN-α、IFN-β表达;动物实验表明90 μg、180 μg的加巴喷丁处理组可减轻RSV感染小鼠的体质量变化、改善肺部病理损伤和降低病毒载量.结论 加巴喷丁可通过抑制病毒复制,调节趋化因子及炎性因子释放,促进干扰素分泌的方式发挥体内抗病毒作用,对RSV感染的C57BL/6小鼠有一定的治疗作用,可改善肺部病理,为进一步临床应用提供实验依据.

To investigate the cause of piglets diarrhea,and the distribution of the serotype and virulence factors of swine Escherichia coli in Beijing,400 diarrhea samples were collected.TSA serum agar culture method was used to isolate Escherichia coli ,serotype identification test and virulence factor genes test were used to verify the presence of O-antigen.64 strains of E .coli were isolated from 400 diarrhea samples,among which 42 strains of E .coli were classified as 8 se-rotypes:O101(18.7％),O64(12.5％),O8 (10.9％),O20 (10.9％),O45 (4.7％),O149 (4.7％), O2(1.6％)and O89(1.6％),and the major virulence factors were STa,Stx2e,astA and eaeA. There were 8 mainly serotypes that caused piglets diarrhea in Beijing area,among which O101 se-rotype accounted for the highest proportion.The major virulence factors were astA and eaeA,ac-counted for more than 50％ of all strains,STa and Stx2e accounted for more than 30％ of all strains.These data would provide effective data to support the prevention and control of piglet di-arrhea in Beijing.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot and mouth disease (HFMD), which erupts in the Asia-Pacific regions. A bivalent vaccine against both EV71 and CVA16 is highly desirable. In the present study, on the bases that an experimental bivalent vaccine comprising of inactivated EV71 and CVA16 induces a balanced protective immunity against both EV71 and CVA16, we compare the immunogenicity and reactogenicity of one fourth of a full dose of an intradermal vaccine administered by needle-free liquid jet injector with a full dose of an intramuscular vaccine administered by needle-syringe in monkeys. The results suggest that intradermal injection of a fractional dose of an inactivated HFMD vaccine elicits similar immunogenicity and reactogenicity to intramuscular inoculation of a full dose of an Al(OH)3-adjuvanted vaccine, regardless of whether monovalent or bivalent vaccines were used. Our results support the use of an intradermal bivalent vaccine strategy for HFMD vaccination in order to satisfy the requirements and reduce the costs.